Many processes in cells are mediated by large protein assemblies or multi-protein complexes that work together in a highly regulated manner to maintain normal homeostasis. Aberrations in multi-subunit protein complexes can lead to various disease states. In order to understand how protein machines function in various cellular processes and to aid future drug development, mapping interaction networks of protein complexes has become one of the major endeavors in modern proteomics research. In order to capture protein interactions of all types in living cells, we have developed the QTAX method to allow effective capture, purification, and quantitative identification of stable, weak and/or transient protein interactions of protein complexes. This strategy allows generating an authentic snapshot of protein interaction networks as they exist in living cells. The ubiquitin-proteasome system (UPS) represents the major pathway for regulated degradation of intracellular proteins in eukaryotes, which helps control numerous essential physiological processes. Aberration of the UPS is known to lead to a variety of human diseases including cancer. The first class of anticancer drugs acts through general inhibition of the UPS and is effective but cannot be used for long-term treatment. This is because general proteasome inhibition will block the entire degradation process and affect many processes nonspecifically. In order to develop more effective and less toxic therapeutics, it is necessary to determine proteasome structural and functional heterogeneity and obtain a better understanding of the molecular mechanisms of UPS regulation and substrate specificity. Toward these goals, this proposal aims to investigate two previously uncharacterized subgroups of human 26S proteasome complexes in mammalian cells and to advance QTAX-based protein interaction study to new levels by defining structural topologies of in vivo protein complexes. The development of these novel strategies will be an exciting technological advancement in proteomics research. The specific aims include: 1) To unravel structural and functional differences of DNA-bound and non-DNA bound human proteasome complexes using the QTAX strategy; 2) To develop the next generation of the QTAX strategy for mass spectrometric characterization of in vivo protein interaction topologies of protein complexes; 3) To determine quantitative differences in ubiquitin receptor- associated proteasome complexes by a split-tag strategy and quantitative mass spectrometry.